Phenotypic profiling of adaptation to oxidative stress

Caloric restriction extends lifespan, an effect once thought to involve attenuation of reactive oxygen species (ROS) generated by aerobic metabolism. However, recent evidence suggests that caloric restriction may in fact raise ROS levels, which in turn provides protection from acute doses of oxidant through a process called adaptation. To shed light on the molecular mechanisms of adaptation, we designed a series of genome-wide deletion fitness screens to identify genes involved in adaptation to hydrogen peroxide. We designed a series of yeast genome-wide phenotyping experiments using the publicly available pool of 4,831 viable single-gene deletion strains. Each strain in the pool incorporates a pair of unique oligonucleotide barcode tags, which allow the relative growth rates of all strains to be tracked in competitive growth experiments by hybridization of pooled genomic DNA to a barcode microarray. We elicited adaptation using a protocol consisting of a mild pretreatment of hydrogen peroxide (0.1 mM H2O2 for 45 min) followed by a later high dose (0.4 mM H2O2 for 1 hr). For purposes of comparison, we also conducted an acute protocol which exposed cells to the high dose only (0.4 mM H2O2 for 1 hr). In a first experiment, two identical pools of deletion mutants were treated with the adapted or acute protocol, respectively, and directly compared on a barcode array (with six biological replicates). In a second experiment, a pool subjected to the acute treatment was compared against an untreated pool (with four biological replicates).